Insulin resistance is important in the development of adult-onset diabetes mellitus and several lines of evidence indicate that an initial defect in adult onset diabetes is reduced insulin-stimulated recruitment of GLUT4 glucose transporter from a sequestered intracellular site to the cell surface in muscle and fat cells. An increase in cell surface (plasma membrane) GLUT4 amount allows for an increased rate of facilitated diffusion of glucose into cells. Presently available methods of determining or measuring GLUT4 translocation, such as methods involving assessment of 3H 2-deoxyglucose uptake, cell fractionation, counting radioactivity that binds to adherent cells on a multiwell plate, plasma membrane sheet assays and immunofluorescence microscopy are laborious and/or only semiquantitative. It would be of considerable interest to be able to measure GLUT4 glucose transporter protein translocation easily and quantitatively.
The present invention relates to a method of assessing translocation of a protein of interest in cells which occurs when the cells are exposed to a condition or treated in such a manner that a protein that is sequestered at an intracellular location moves (is translocated) to the cell surface (plasma membrane) or, alternatively, a protein at the cell surface moves (is translocated) to an intracellular location. It also relates to a method of determining whether a condition or set of conditions to which cells are exposed or contacted alters (induces, enhances or inhibits) translocation of a protein of interest known to undergo translocation under certain conditions. The protein whose movement is being assessed is referred to as a protein of interest. The method is carried out using a modified form of the protein of interest, referred to as a modified protein of interest or a reporter protein. The reporter protein is the protein of interest modified in such a manner that it comprises one or more tags that allow quantification of relative amounts of the protein that are at the cell surface and of the total amounts of the protein in the cells. For example, it can comprise one or more intracellular tag(s) and one or more extracellular tag(s). The extracellular tag(s) and the intracellular tag(s) are distinct tags; that is, they are different tags that can be detected separately/distinguished from one another (e.g., two fluorescent tags detectable at different wavelengths). The reporter protein moves within the cells in essentially the same way as the protein of interest. The intracellular tag e.g., an intracellular fluorescent tag corresponds to the total protein of interest in the cell and does not change in quantity depending on the location of the reporter protein in the cell. The intracellular tag is fused in-frame in the DNA or RNA sequence that encodes the protein. The extracellular tag (e.g., an epitope(s) or radioactive label) is detectable only if the tag is extracellular, since the detector used is one that is unable to (does not) cross the cell membrane; the detector can be, for example, an antibody or other compound that recognizes (binds) the extracellular tag. Thus, the extent to which the extracellular tag is detectable (e.g., the intensity of fluorescence due to the epitope tag(s)) is indicative of the extent to which the protein of interest is present at the cell membrane. The intracellular tag can be any fluorescent tag, such as Green Fluorescent Protein (GFP), Blue Fluorescent Protein (BFP), Red Fluorescent Protein (RFP) or a sequence of amino acids designed to bind a molecule that has fluorescent, radioactive, or other detectable characteristics. The extracellular tag is generally an epitope tag or tags that is recognized by an antibody. The antibody can be monoclonal or polyclonal and can itself be detectably labeled (e.g., fluorescently labeled) or can be recognized (bound) by a detectably labeled antibody (a labeled secondary antibody). For example, the epitope tag(s) can be recognized by a primary or secondary antibody labeled with phycoerythrin (PE). Any pair of fluorescent labels can be used, as long as they are detectable at distinct wavelengths (different wavelengths). If there are two or more intracellular tags and/or two or more extracellular tags, it is only necessary that there are distinct detectable differences (such as distinct/different wavelengths) that correspond to surface protein of interest and total protein of interest. Alternatively radioactive, colorimetric, luminescent or other detection strategies may be used. The only requirements are that 1) the two tags can be detected individually and each can be quantified and 2) the addition of tag(s) does not substantially alter trafficking of the protein of interest with cells.
The extent of translocation of the protein of interest is assessed by culturing or treating cells that contain the reporter protein (referred to as test cells) under a condition(s) to be assessed for their effects on translocation of the protein of interest to the cell membrane; determining the intensity of the intracellular tag (e.g., intensity of fluorescence of an intracellular tag, such as GFP, RFP or BFP) and the intensity of the extracellular tag (e.g., by measuring the intensity of fluorescence of epitope tag(s) by means of fluorescently labeled antibodies); calculating the proportion or fraction of and determining a value corresponding to the total cellular modified protein of interest that is present at the cell surface and comparing the resulting proportion with the corresponding proportion calculated for control cells. Control cells are the same type of cells as the test cells and are cultured or handled in the same manner as are the test cells, except that control cells are not cultured or treated under the conditions being assessed for their effects on translocation to the cell membrane. For example, if test cells are cultured in the presence of a hormone or growth factor, control cells are cultured under the same conditions as the test cells, except in the absence of the hormone or growth factor. Two measurements (such as fluorescence at two different wavelengths), F1 and F2, correspond, respectively, to cell surface protein of interest and total protein of interest in the cells. If the proportion of protein of interest at the cell membrane to total protein of interest is greater for test cells than for control cells, translocation has occurred in the test cells and the condition under which the test cells were cultured or treated (e.g., in the presence of a hormone or growth factor) is a condition that causes or enhances translocation from an intracellular location to the cell membrane. Translocation from the cell membrane to an intracellular location for a protein of interest can also be assessed using the modified protein of interest and the method of the present invention.
In a particular embodiment, the protein whose translocation is assessed is GLUT4 and the modified protein of interest is modified GLUT4, also referred to as GLUT4 reporter, which includes an intracellular tag and at least one, and preferably multiple, extracellular tag(s). For example, the intracellular tag is GFP, RFP or BFP and the extracellular tag(s) are epitope tag(s), which can be detected using a fluorescent or a radioactive label. The ratio of protein of interest at the cell membrane (F1) to total protein of interest in test cells (F2) is compared to the ratio of protein of interest at the cell membrane (F1) to total protein of interest in the control cells, (F2). If the proportion is greater in the test cells is greater than the control cells, the condition(s) is an enhancer of/causes translocation to the cell membrane.
As described below with specific reference to GLUT4, preferably three sets of values, each consisting of fluorescence intensities at two distinct wavelengths, are determined in the method of assessing change in the proportion of the protein of interest that is present at the plasma membrane (and, thus, the extent to which translocation has occurred). The two wavelengths, F1 and F2, correspond, respectively, to cell surface (plasma membrane) protein of interest and total protein of interest in the cell. The three sets of values are F1 and F2 in control cells, F1 and F2 in background cells and F1 and F2 in test cells. In some cases, separate background sets of values may be required for tests and for control cells so that there are four sets of values in all. Cell surface protein of interest can be measured by a fluorophore (e.g., PE) coupled to an antibody that binds to externalized epitope tag(s), such as myc epitope tag(s), but not to intracellular myc epitope tag, or by a secondary fluorophore-bearing antibody that recognizes a primary antibody that recognizes the myc epitope. The three sets of values are as described below with specific reference to GLUT4.
Insulin stimulates glucose uptake in muscle and adipose tissues by causing translocation of the GLUT4 glucose transporter from a sequestered, intracellular compartment to the plasma membrane. Described herein is a novel assay to measure changes in the proportion (fraction) of total GLUT4 present at the plasma membrane of cultured cells expressing a reporter protein, based on measurement of a detectable tag corresponding to cell surface amount of GLUT4 and measurement of a second (different) detectable tag corresponding to total amount of GLUT4 in the cells. The two tags can both be, for example, fluorescent tags that are detectable at different wavelengths or two different types of tags, such as a fluorescent tag corresponding to total cell GLUT4 and a radioactive label or an epitope(s) corresponding to GLUT4 at the cell surfaces. Various types of measurement can be used to detect the two tags. For example, if fluorescence is used to detect the two tags, then flow cytometry can be used to measure the tags quantitatively and on a cell-by-cell basis. This assay has been used to demonstrate that insulin stimulates GLUT4 translocation with identical kinetics at all times during differentiation of a subline of 3T3-L1 adipocytes, even in undifferentiated, confluent fibroblasts. This effect is invariably blocked by phosphatidylinositol-3-kinase inhibitors. In all cases, the reporter is reinternalized after insulin removal, and recycles upon insulin readdition.
As also described herein, insulin has been shown to trigger GLUT4 externalization in Chinese hamster ovary (CHO) cells, with initial kinetics and magnitude identical to those in 3T3-L1 adipocytes, when the CHO cells are cultured under the same conditions as those under which the 3T3-L1 adipocytes are cultured. Thus, CHO cells, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes all contain an intracellular recycling compartment to which GLUT4 is targeted, and that is rapidly mobilized upon insulin addition.
Described herein is an assay for measuring the effects of conditions on translocation (movement of a protein of interest from an intracellular location to the plasma membrane or from the plasma membrane to an intracellular location). In a specific embodiment, the invention is an assay for measuring the effects of conditions, such as the presence of insulin and other stimulators, on targeting of GLUT4 to the plasma membrane of cells and, thus, for measuring their effects on glucose uptake. Also described herein is a method of assessing the effect of a drug on translocation of a protein of interest, such as GLUT4, from an internal (intracellular) location to the plasma membrane or from the plasma membrane to an intracellular location. The method is useful to identify drugs which alter translocation of the protein of interest. Of particular interest is a method of identifying drugs that alter GLUT4 translocation, such as drugs that increase the fraction of GLUT4 in the plasma membrane (in the absence and/or presence of insulin) or that enhance the effect of insulin on GLUT4 translocation to the plasma membrane and, thus, are useful in enhancing uptake of glucose into cells, since stimulation of glucose transport by insulin requires translocation of GLUT4 to the plasma membrane. An increase in GLUT4 at the cell membrane allows for a rapid increase in the rate of facilitated diffusion of glucose into the cell. Such drugs are useful, for example, in treating insulin resistance, such as occurs in adult-onset diabetes, obesity and polycystic ovary syndrome. Modified protein of interest; DNA encoding modified protein of interest; expression vectors, cells and cell lines containing DNA encoding modified protein of interest; and expression vectors, cells and cell lines expressing DNA encoding modified protein of interest are also described. Modified GLUT4 glucose transporter protein; DNA encoding modified GLUT4; expression vectors (e.g., retroviral vectors, such as replication-deficient retroviral vectors, adenoviral vectors and other expression vectors for eukaryotic cells), cells and cell lines containing DNA modified GLUT4; and expression vectors and cells and cell lines expressing modified GLUT4 are particular embodiments of this invention. They are useful in the methods described herein.
Described herein are the production of a DNA construct encoding one embodiment of modified GLUT4 and cells in which modified GLUT4 was expressed. Modified GLUT4 is GLUT4 protein which comprises at least one (one or more) detectable tag, such as at least one epitope tag in an extracellular domain and a detectable tag, such as a fluorescent tag, in an intracellular domain. The intracellular tag is fused in-frame in the modified GLUT4 protein. The epitope tag is, for example, present in the first extracellular domain of GLUT4 and is, for example, a myc epitope or myc epitopes. The fluorescent tag is, for example, GFP or BFP. In one embodiment of the construct described herein, GFP provides an internal control for the amount of protein present. For example, the externalized epitope tag is detected with a xe2x80x9credxe2x80x9d fluorescent antibody and GFP is detected using xe2x80x9cgreenxe2x80x9d fluorescence. Translocation can, as a result, be measured by the increase in the ratio of xe2x80x9credxe2x80x9d fluorescence to xe2x80x9cgreenxe2x80x9d fluorescence. This ratio can be measured, for example, using flow cytometry or a fluorescent plate reader. Translocation of modified GLUT4 is detected using an antibody which binds the epitope tag; the bound antibody can be identified by means of a labeled (e.g., fluorescently labeled) second antibody which binds the first antibody. Alternatively, the epitope binding antibody can itself be labeled, such as fluorescently labeled. In either case, detection of the fluorescent label is carried out using known methods, such as flow cytometry, fluorescent plate reader or microscopy. The eukaryotic (e.g., mammalian, avian, reptilian) cells in which modified GLUT4 is expressed can be primary cells or a cell line, such as mouse, human, hamster, rat, rabbit, guinea pig, monkey, dog, cat or human primary cells or cell lines derived from one of these sources.